Opiate analgesics act at the mu opioid receptor (MOR) in humans to alleviate pain. However, this receptor is also the means by which these drugs produce unwanted effects such as constipation, dependence and addiction. The overall potency and efficacy of an agonist at the receptor may be determined not only by how well the drug binds the receptor but also by how well the receptor engages with intracellular signaling proteins, such as barrestin2. Our studies over the last decade have led us to hypothesize that if a drug could activate the MOR yet not induce barrestin2 interactions with the receptor, then such a drug might be an efficacious analgesic with limited side effects, producing less tolerance, dependence and constipation. Based on our extensive studies of barrestin regulation of MOR, we believe that the ideal opioid agonist would have little to no efficacy for recruiting barrestin2 while having full efficacy for G protein coupling or other cellular signaling pathways. In this proposal, we will pharmacologically characterize and optimize compounds from a small molecule library representing four distinct scaffolds. These compounds have been synthesized by Dr. Thomas Bannister, an experienced medicinal chemist at Scripps Florida who has previously worked in the pharmaceutical industry generating, among many drug candidates, analgesics based on fentanyl. Our preliminary data shows that we have agonists that possess partial to full efficacy (relative to the enkephalin analog, DAMGO) in G protein coupling, ERK activation, and cellular impedance assays. Remarkably, most of these analogs do not recruit barrestin2, even under conditions that favor MOR-barrestin interactions. In this multidisciplinary study, the Bohn pharmacology laboratory will work in a highly collaborative manner with the Bannister medicinal chemistry laboratory to generate and optimize multiple derivatives on these 4 scaffolds (Aim 1). We will use several cell-based assays to characterize the signaling parameters induced by these compounds with the goal of finding opiates that possess high efficacy in signaling cascades yet produce no barrestin2 coupling to MOR (Aim 2). Such compounds will be tested in mouse models to determine if their signaling properties correlate with their ability to produce analgesia with fewer side effects (Aim 3). Finally, in collaboration with Dr. Michael Cameron of Scripps Florida, we will evaluate the DMPK properties of our best candidate compounds. The information garnered from this proposal will prove useful in the future development of clinical pain relievers with limited side effects.